Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

Abstract

Proliferating cell nuclear antigen (PCNA) coordinates multienzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the interdomain connector loop (IDCL) of PCNA through its PCNA‐interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N‐terminal deletions to establish that DNA binding and nick‐sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box–IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. As the rate constants for single‐turnover ligation for the full‐length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C‐terminal mutants of PCNA are not able to stimulate nick‐sealing activity of Entamoeba histolytica DNA ligase I. Our data support the notion that the C‐terminal region of PCNA may be involved in promoting an allosteric transition in E. histolytica DNA ligase I from a spread‐shaped to a ring‐shaped structure. This study suggests that the ring‐shaped PCNA is a binding platform able to stabilize coevolved protein–protein interactions, in this case an interaction with DNA ligase I.

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica

“…After 15–30 min of phagocytosis, all four proteins appeared spread in the cytoplasm and in the membranes and lumen of phagosomes and surrounding the ingested erythrocytes (Figure 2C ). Intriguingly, EhVps24 and EhVps2, consistently migrated to the nuclei at longer times of phagocytosis (60 min), co-localizing with the nuclear protein pCNA (Trasviña-Arenas et al, 2017 ) and DAPI staining (Figures 2C,E ). Altogether, these results demonstrated that the ESCRT-III orthologs associate to erythrocytes and erythrocyte-containing phagosomes, suggesting that ESCRT-III proteins are a part of the machinery involved in phagocytosis.…”

“…In the case of nuclear co-localization, rEhVps2 and EhVps24 were detected by secondary antibodies as described previously. pCNA (Trasviña-Arenas et al, 2017 ) protein was detected incubating cells with α-pCNA (Trasviña-Arenas et al, 2017 ) (1:200) for 1h at 37°C, followed by incubation with α-mouse TRITC-labeled antibodies (1:100) at 37°C for 1h. Nuclei were counterstained by 2.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min.…”

The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair

“…In several systems, DNA ligase I is recruited to DNA damaged sites by its interaction with PCNA (Mortusewicz et al, 2006 ). Moreover, EhDNAligI activity is enhanced by EhPCNA and these proteins present a functional interaction in vitro (Cardona-Felix et al, 2011 ; Trasviña-Arenas et al, 2017 ). Thus, we tested whether during recovery from UV and H 2 O 2 treatments, EhDNAligI co-localizes with EhPCNA using an antibody generated against the rEhDNAligI recombinant protein (Cardona-Felix et al, 2010 , 2011 ).…”

Intestinal tract and parenteral multi-organ sequential pathological injury caused by necrotizing enterocolitis

“…At present, PCNA has been widely used in the study of cell proliferation kinetics [16]. In this study, we found that lung, liver and kidney cells proliferated to repair their corresponding organs after injury, but the duration of proliferation and degree of each organ were also different due to their differing abilities to self-repair and mechanism of each organ.…”